Mikkola I, Bruun J A, Holm T, Johansen T
Department of Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway.
J Biol Chem. 2001 Feb 9;276(6):4109-18. doi: 10.1074/jbc.M008882200. Epub 2000 Nov 7.
The Pax6 genes encode evolutionary conserved transcription factors that act high up in the regulatory hierarchy controlling development of central organs such as the eyes and the central nervous system. These proteins contain two DNA-binding domains. The N-terminal paired domain is separated from a paired-type homeodomain by a linker region, and a transactivation domain is located C-terminal to the homeodomain. Vertebrate Pax6 genes express a paired-less isoform of Pax6 (Pax6DeltaPD) from an internal start codon in the coding region between the paired domain and homeodomain. We now provide evidence for an interaction between the full-length isoform and Pax6DeltaPD, which enhances the transactivation activity of Pax6 from paired domain-binding sites. The paired-like homeodomain protein Rax behaved similarly to Pax6DeltaPD. Both Pax6DeltaPD and Rax bound to the homeodomain of Pax6 in vitro in the absence of specific DNA binding. Coimmunoprecipitation experiments following cotransfection confirmed the existence of complexes between Pax6 and Pax6DeltaPD, Pax6 and Rax, and Pax6DeltaPD and Rax in vivo. Interestingly, the C-terminal subdomain of the paired domain and the homeodomain can interact with each other. The paired domain can also interact with itself. Surprisingly, GST pull-down assays revealed that the homeodomains of such diverse proteins as Chx10, Six3, Lhx2, En-1, Prep1, Prox1, and HoxB1 could all bind to Pax6, and several of these enhanced Pax6-mediated transactivation upon coexpression. Since many homeodomain proteins are coexpressed with Pax6 in several tissues during development, our results indicate the existence of novel regulatory interactions that may be important for fine tuning of gene regulation.
Pax6基因编码进化上保守的转录因子，这些转录因子在控制眼睛和中枢神经系统等中央器官发育的调控层级中处于较高位置发挥作用。这些蛋白质包含两个DNA结合结构域。N端的配对结构域通过一个连接区与配对型同源结构域分开，一个反式激活结构域位于同源结构域的C端。脊椎动物的Pax6基因从配对结构域和同源结构域之间编码区的一个内部起始密码子表达一种无配对结构域的Pax6异构体（Pax6DeltaPD）。我们现在提供了全长异构体与Pax6DeltaPD之间相互作用的证据，这种相互作用增强了Pax从配对结构域结合位点的反式激活活性。配对样同源结构域蛋白Rax的行为与Pax6DeltaPD相似。在没有特异性DNA结合的情况下，Pax6DeltaPD和Rax在体外均与Pax6的同源结构域结合。共转染后的免疫共沉淀实验证实了Pax6与Pax6DeltaPD、Pax6与Rax以及Pax6DeltaPD与Rax在体内存在复合物。有趣的是，配对结构域的C端亚结构域和同源结构域可以相互作用。配对结构域也可以自身相互作用。令人惊讶的是，GST下拉实验表明，诸如Chx10、Six3、Lhx2、En-1、Prep1、Prox1和HoxB1等多种不同蛋白质的同源结构域都能与Pax6结合，其中几种在共表达时增强了Pax6介导的反式激活。由于许多同源结构域蛋白在发育过程中与Pax6在多个组织中共表达，我们的结果表明存在可能对基因调控的精细调节很重要的新型调控相互作用。
